Differential expression of the Trichoderma reesei beta-xylanase II (xyn2) gene in the xylose-fermenting yeast Pichia stipitis.

Abstract

The transcriptional control of two native promoters and one heterologous promoter and the production of a heterologous protein from these promoters were evaluated in the xylose-fermenting yeast Pichia stipitis cultivated on xylose and glucose as carbon sources, using the beta-xylanase II xyn2 gene of Trichoderma reesei. The xyn2 gene open reading frame was fused to the P. stipitis xylose reductase gene (XYL1) promoter, the P. stipitis transketolase gene (TKL) promoter and the Saccharomyces cerevisiae phosphoglycerate kinase gene (PGKI) promoter DNA sequences on episomal plasmids. The plasmids were transformed into Pichia stipitis and gene expression and beta-xylanase production monitored. The XYL1 promoter was shown to be inducible in the presence of xylose, as xyn2 transcription and beta-xylanase activity could be measured when the recombinant strain was cultivated on xylose but not when it was cultivated on glucose. TKL promoter expression was found to be constitutive when either glucose or xylose was used as sole carbon source. The PGK1 promoter did not promote xyn2 transcription in P. stipitis. The molecular size of the recombinant Xyn2 protein produced by P. stipitis was 20.7 kDa, which is similar to that of the native T. reesei Xyn2 protein. This indicates no or minimal glycosylation of the recombinant protein. The recombinant xyn2-expressing strain also yielded twice the amount of biomass yielded by the control strain when cultivated in medium containing 1% birchwood xylan as sole carbon source.